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Telomere shortening limits the proliferative capacity of a cell, but perhaps surprisingly, shortening is also known to be associated with increased rates of tumor initiation. A current hypothesis suggests that telomere dysfunction increases tumor initiation by induction of chromosomal instability, but that initiated tumors need to reactivate telomerase for genome stabilization and tumor progression. This concept has not been tested in vivo, since appropriate mouse models were lacking. Here, we analyzed hepatocarcinogenesis in a mouse model of inducible telomere dysfunction on a telomerase-proficient background, in telomerase knockout mice with chronic telomere dysfunction (G3 mTerc-/-), and in WT mice with functional telomeres and telomerase. Transient or chronic telomere dysfunction enhanced the rates of chromosomal aberrations during hepatocarcinogenesis, but only telomerase-proficient mice exhibited significantly increased rates of macroscopic tumor formation in response to telomere dysfunction. In contrast, telomere dysfunction resulted in pronounced accumulation of DNA damage, cell-cycle arrest, and apoptosis in telomerase-deficient liver tumors. Together, these data provide in vivo evidence that transient telomere dysfunction during early or late stages of tumorigenesis promotes chromosomal instability and carcinogenesis in telomerase-proficient mice.

The tumour suppressor p53 activates Puma-dependent apoptosis and p21-dependent cell-cycle arrest in response to DNA damage. Deletion of p21 improved stem-cell function and organ maintenance in progeroid mice with dysfunctional telomeres, but the function of Puma has not been investigated in this context. Here we show that deletion of Puma improves stem- and progenitor-cell function, organ maintenance and lifespan of telomere-dysfunctional mice. Puma deletion impairs the clearance of stem and progenitor cells that have accumulated DNA damage as a consequence of critically short telomeres. However, further accumulation of DNA damage in these rescued progenitor cells leads to increasing activation of p21. RNA interference experiments show that upregulation of p21 limits proliferation and evolution of chromosomal imbalances of Puma-deficient stem and progenitor cells with dysfunctional telomeres. These results provide experimental evidence that p53-dependent apoptosis and cell-cycle arrest act in cooperating checkpoints limiting tissue maintenance and evolution of chromosomal instability at stem- and progenitor-cell levels in response to telomere dysfunction. Selective inhibition of Puma-dependent apoptosis can result in temporary improvements in maintenance of telomere-dysfunctional organs.

International audience; BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc(-/-)) with short telomeres compared to wild type mice (mTerc(+/+)) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc(-/-) mice compared to mTerc(+/+) mice. Seven days after chemical induced damage, G3 mTerc(-/-) mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc(+/+) mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people. ispartof: PLoS One vol:6 issue:11 ispartof: location:United States status: published

Telomere shortening and inactivation of cell cycle checkpoints characterize carcinogenesis. Whether these molecular features coincide at specific stages of human hepatocarcinogenesis is unknown. The preneoplasia–carcinoma sequence of human HCC is not well defined. Small cell changes (SCC) and large cell changes (LCC) are potential precursor lesions. We analyzed hepatocellular telomere length, the prevalence of DNA damage, and the expression of p21 and p16 in biopsy specimens of patients with chronic liver disease (n = 27) that showed different precursor lesions and/or HCC: liver cirrhosis (n = 25), LCC (n = 26), SCC (n = 13), and HCC (n = 13). The study shows a decrease in telomere length in nondysplastic cirrhotic liver compared with normal liver and a further significant shortening of telomeres in LCC, SCC, and HCC. HCC had the shortest telomeres, followed by SCC and LCC. Hepatocytes showed an increased p21 labeling index (p21-LI) at the cirrhosis stage, which remained elevated in most LCC. In contrast, most SCC and HCC showed a strongly reduced p21-LI. Similarly, p16 was strongly expressed in LCC but reduced in SCC and not detectable in HCC. γH2AX-DNA-damage-foci were not detected in LCC but were present in SCC and more frequently in HCC. These data indicate that LCC and SCC represent clonal expansions of hepatocytes with shortened telomeres. Conclusion: The inactivation of cell cycle checkpoints coincides with further telomere shortening and an accumulation of DNA damage in SCC and HCC, suggesting that SCC represent more advanced precursor lesions compared with LCC. (HEPATOLOGY 2007;45:968–976.)

Telomere shortening limits the proliferative lifespan of human cells by activation of DNA damage pathways, including upregulation of the cell cycle inhibitor p21 (encoded by Cdkn1a, also known as Cip1 and Waf1)) (refs. 1-5). Telomere shortening in response to mutation of the gene encoding telomerase is associated with impaired organ maintenance and shortened lifespan in humans and in mice. The in vivo function of p21 in the context of telomere dysfunction is unknown. Here we show that deletion of p21 prolongs the lifespan of telomerase-deficient mice with dysfunctional telomeres. p21 deletion improved hematolymphopoiesis and the maintenance of intestinal epithelia without rescuing telomere function. Moreover, deletion of p21 rescued proliferation of intestinal progenitor cells and improved the repopulation capacity and self-renewal of hematopoietic stem cells from mice with dysfunctional telomeres. In these mice, apoptotic responses remained intact, and p21 deletion did not accelerate chromosomal instability or cancer formation. This study provides experimental evidence that telomere dysfunction induces p21-dependent checkpoints in vivo that can limit longevity at the organismal level.

Telomeres form the ends of eukoryotic chromosomes. The main function of telomeres is to stabilise chromosome ends thus to prevent chromosomal instability (CIS) and activation of DNA damage response. Telomere shortening due to the end replication problem of DNA polymerase limits the proliferative capacity of human cells to 50–70 cell doublings. The holoenzyme telomerase prevents ongoing telomere shortening by de novo synthesis of telomere repeats. However, in humans, postnatal telomerase expression is suppressed in most somatic tissues—including the liver. Telomere shortening limits the regenerative capacity of hepatocytes during chronic liver disease and hepatocellular senescence is associated with cirrhosis formation. In animal models, it has been shown that telomerase reactivation can improve liver regeneration and prevent cirrhosis formation induced by telomere shortening. The future use of telomerase activation for treatment of chronic liver disease depends on its effect on hepatocarcinogenesis. Telomerase reactivation appears to be required for hepatocellular carcinoma progression whereas telomere shortening is linked to CIS and tumor initiation. This review will focus on the role of telomere shortening and telomerase activation in cirrhosis and hepatocarcinogenesis and the potential use of telomerase activators or inhibitors for these disease stages.

Background & Aims p53 limits the self-renewal of stem cells from various tissues. Loss of p53, in combination with other oncogenic events, results in aberrant self-renewal and transformation of progenitor cells. It is not known whether loss of p53 is sufficient to induce tumor formation in liver. Methods We used AlfpCre mice to create mice with liver-specific disruption of Trp53 ( AlfpCre + Trp53 Δ2-10/Δ2-10 mice). We analyzed colony formation and genomic features and gene expression patterns in liver cells during hepatocarcinogenesis in mice with homozygous, heterozygous, and no disruption of Trp53. Results Liver-specific disruption of Trp53 consistently induced formation of liver carcinomas that had bilineal differentiation. In nontransformed liver cells and cultured primary liver cells, loss of p53 (but not p21) resulted in chromosomal imbalances and increased clonogenic capacity of liver progenitor cells (LPCs) and hepatocytes. Primary cultures of hepatocytes and LPCs from AlfpCre + Trp53 Δ2-10/Δ2-10 mice, but not Cdkn1a −/− mice, formed tumors with bilineal differentiation when transplanted into immunocompromised mice. Spontaneous liver tumors that developed in AlfpCre + Trp53 Δ2-10/Δ2-10 mice had significant but complex alterations in expression of Rb checkpoint genes compared with chemically induced liver tumors that developed mice with wild-type Trp53. Conclusions Deletion of p53 from livers of mice is sufficient to induce tumor formation. The tumors have bilineal differentiation and dysregulation of Rb checkpoint genes.

Introduction: A current concept of carcinogenesis indicates that telomere dysfunction can increase tumor initiation by induction of chromosomal instability, but initiated tumor cells need to reactivate telomerase for genome stabilization and tumor progression. So far, this concept has not been proven in vivo since appropriate mouse models were lacking. Methods: Here, we analyzed hepatocarcinogenesis (i) in a novel mouse model of inducible telomere dysfunction on a telomerase-proficient background, (ii) in telomerase knockout mice with chronic telomere dysfunction (G3 mTerc-/-), and (iii) in wild-type mice with functional telomeres and telomerase. Results: Transient or chronic telomere dysfunction enhanced the rates of chromosomal aberrations during hepatocarcinogenesis, but only telomerase-proficient mice exhibited significantly increased rates of macroscopic tumor formation and cancer cell proliferation in response to telomere dysfunction. In contrast, telomere dysfunction resulted in pronounced accumulation of DNA damage, cell cycle arrest and apoptosis in telomerase-deficient liver tumors. Conclusion: Together, these data provide the first in vivo evidence that telomere dysfunction can promote chromosomal instability and carcinogenesis in telomerase-proficient mice in the absence of additional, genetic checkpoint defects at germline level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4811. doi:1538-7445.AM2012-4811

Cytogenetic analysis of chromosomes using Fluorescence in situ Hybridization (FISH) is a powerful method for the study of chromosome structure, organization, and stability. However, to date, the utility of FISH has largely depended upon access to cloned template DNA used to generate probes. Clones for specific regions are often unavailable, or the genomic template may contain repetitive and other non-informative sequences which can confound FISH analysis. We have leveraged our ability to chemically synthesize oligonucleotides in massively parallel reactions to produce DNA libraries that can be used to generate FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to target only the most informative elements in the genomic region to be analyzed and avoiding any repetitive elements or regions homologous to other non-targeted loci. Our FISH protocol is similar to ones used for FISH with BAC probes. Using our methods, human chromosomal regions as small as 1.8 kb and as large as chromosome arms were successfully visualized in both metaphase and interphase cells. Using our probe design methods, we readily obtained specific and robust signals for chromosomal regions rich in repeats and/or GC content. We also investigated the utility of these probes for studies of mouse chromosomes by generating probes for over ten loci in the mouse genome. All the probes hybridized well using cytologic and cryosectioned samples. In addition, FFPE samples showed promising preliminary results. We have also applied our FISH methods to develop chromosome-specific enumeration probes for the mouse genome at pericentromeric regions. These enumeration probes should be useful for detecting aneuploidies, polyploidies, and other genomic rearrangements that were previously difficult to study at single-cell resolution, particularly in tissue samples. Successful co-hybridization of chromosome enumeration probes and loci-specific probes in mouse intestinal crypt cells allowed for accurate copy number analyses in tissue samples. We are also continuing to explore other breakthrough applications that are enabled by our oligonucleotide-based FISH methodology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 328.

Background: Defects in the p53-signaling checkpoint have been described in 30-60% of human hepatocellular carcinoma (HCC) and are associated with less differentiated tumors and poor prognosis. The contribution of p53 deletion by itself to hepatocarcinogenesis is not known. Furthermore it is not clear, if liver stem cells play a role in the onset of liver tumor formation. Methods: We generated mice with liver specific TRP53 deletion (TRP53−/−) by crossing conditional TRP53 KO mice with AlfpCRE transgenic mice (CRE expression under the albumin-alphafetoprotein-enhancer-promoter). In vitro cultures were established from the developing liver tumors and analyzed for potential tumor stem cells using xenotransplantation. Endogenous liver stem/progenitor cells were analysed using FACS purification and in vitro culturing of purified liver stem cells (CD13+CD133+CD49f+). Results: Mice with liver specific TRP53 deletion developed liver tumors with a mixed differentiation (hepatocellular and cholangiocellular carcinoma). Purification and xenotransplantation of subpopulations of tumor cells from primary tumor cell cultures (using following surface marker: CD13, CD90, CD133, or cKit) revealed no differences in tumor forming capacity in NMRI nu/nu mice. Moreover, tumor formation was seen after xenotransplantation of very small tumor cell numbers (2-cells) indicating that the tumors have an inherent high tumor forming capacity. An analysis of the colony formation capacity of endogenous liver stem/progenitor cells revealed a prolonged survival of bipotent liver stem cells in aged TRP53−/− mice compared to TRP53+/− or TRP53+/+ mice. The last two groups showed a complete loss of bipotent liver stem cells by 8 month of age, whereas bipotent liver stem cells were detected with a frequency of 0,4% in liver of 8-10 month old TRP53−/− mice. Conclusion: This study provides the first experimental evidence that TRP53 deletion leads to a prolonged survival of bipotent liver stem cells and the formation of bi-differentiated liver tumors. These data indicate that the p53-dependent tumor suppressor functions include the restriction of proliferation and survival of bipotent stem cells in the liver. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4242.

Background: Colorectal cancer is after lung cancer the second biggest cancer threat for humans and the tumor burden is rising steadily during human ageing. One important issue during human ageing is the accumulation of DNA damage inducing a p53 dependent response leading to p21-dependent senescence or apoptosis. It has been shown that deletion of DNA repair proteins as well as other components of the DNA damage pathway leads to tumor formation. Methods: In this approach we investigated the role of the tumor suppressor p53 with and without telomere dysfunction in intestinal tumor formation. Late generation of iG4 mTerc−/−; p53lox/lox and iF1 mTerc+/− p53lox/lox mice were crossed to Villin-Cre-ERT2 transgenic mouse model. In 1 month old mice Tamoxifen was injected on 5 consecutive days leading to functional deletion of the Trp53 gene. Results: In aging IF1 Terc+/− p53 deletion did not induce an obvious intestinal phenotype including tumor formation until 24 months. In contrast to p21 deletion the deletion of p53 significantly shortened the lifespan of iG4 Terc−/− Trp53−/− animals compared to iG4 Terc−/− Trp53+/+. This reduction correlated with earlier appearance of crypt atrophy and weight loss but was not associated with tumor formation. Deletion of the Trp53 gene resulted in an abrogation of the p21-dependent senescence and accumulation of DNA damage in the intestinal epithelium of both p53 deleted cohorts. Moreover this correlated with an accumulation of intestinal stem cells carrying high levels of DNA damage. On contrary to p21 deletion the deletion of p53 impaired the depletion of chromosomal instable intestinal stem cells in aging telomere dysfunctional mice. Conclusion: These results provide first proof that deletion of p53 is not able to promote tumorigenesis in the intestinal epithelium of mice and that p53 induction has a protective role preventing accumulation of damaged intestinal stem cells in aging telomere dysfunctional mice. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2972.

Background: Chromosomal instability, short telomeres, and telomerase activation are hallmarks of human hepatocellular carcinoma. Studies in mouse models have shown that telomere dysfunction induces chromosomal instability and tumor initiation. However, telomere stabilization is required for tumor progression since too high levels of instability impair tumor cell survival. These results suggested that a sequence of telomere dysfunction followed by telomerase re-activation may enhance tumor initiation and progression during human carcinogenesis. Aims: The aim of this study was to test the hypothesis that telomere dysfunction followed by telomerase activation promote tumorigenesis. Methods: We produced transgenic mice allowing doxycycline inducible and transient expression of TRF2DBDM (inducing a transient peak of telomere dysfunction) in the liver. These mice were injected at postnatal day 15 with di-ethyl-nitrosamine. The same mice were treated with intrasplenic injection of doxycycline to induce transient expression of TRF2DBDM and telomere dysfunction. Control mice lacked the TRF2DBDM transgene but were treated the same way. The animals were analysed at an age of 13 months. Results: Doxycycline induced expression of TRF2DBDM in the liver of double-transgenic mice correlated with transient induction of telomere dysfunction in 2−4 month old mice as measured by induction of anaphase bridges (morphological correlate of chromosomal fusions). After 13 months both the LAPrtTA-TRF2DBDM (n = 23) and the control group (n = 29) developed HCC. However, transient telomere dysfunction in LAPrtTA-TRF2DBDM double transgenic mice strongly increased tumor formation. The tumor size was significantly increased by more than two fold in male LAPrtTA-TRF2DBDM mice compared to controls (p< 0.0001). In addition, the number of tumors was significantly increased in female LAPrtTA-TRF2DBDM mice compared to controls (p = 0.019). Chromosomal instability measured by array CGH revealed an increase of aberrations in double-transgenic mice. Conclusions: The results provide first experimental evidence that transient induction of telomere dysfunction during early stages of hepatocarcinogenesis leads to a significant acceleration of tumor formation. These results appear to be important for human hepatocarcinogenesis, which is characterized by telomere shortening and chromosomal instability.